Dolastatin 15 derivatives

ABSTRACT

Compounds of the present invention include cell growth inhibitors which are peptides of Formula I, 
       A-B-D-E-F-(G) r -(K) s -L  (I),
 
     and acid salts thereof, wherein A, B, D, E, F, G and K are α-amino acid residues, and s and r are each, independently,  0  or  1 . L is a monovalent radical, such as, for example, an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group. The present invention also includes a method for treating cancer in a mammal, such as a human, comprising administering to the mammal an effective amount of a compound of Formula I in a pharmaceutically acceptable composition.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/648,446, filed Dec. 29, 2009, which is a continuation of U.S. application Ser. No. 11/406,512 filed Apr. 18, 2006, which is a continuation of U.S. application Ser. No. 10/255,118 filed Sep. 25, 2002, which is a continuation of U.S. application Ser. No. 09/618,694 filed Jul. 18, 2000, which is a continuation of U.S. application Ser. No. 08/896,394 filed Jul. 18, 1997, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A number of short peptides with significant activity as inhibitors of cell growth have been isolated from the Indian Ocean sea hare Dolabella auricularia (Bai et al., Biochem. Pharmacolog_v, 40: 1859-1864 (1990); Beckwith et al., J. Natl. Cancer Inst., 85: 483-488 (1993) and references cited therein). These include Dolastatins 1-10 (U.S. Pat. No. 4,816,444, issued to Pettit et al) and Dolastatin-15 (European Patent Application No. 398558). Dolastatin 15, for example, markedly inhibits the growth of the National Cancer Institute's P388 lymphocytic leukemia (PS system) cell line, a strong predictor of efficacy against various types of human malignancies.

The exceedingly small amounts of the various Dolastin peptides present in Dolahella auricularia (about 1 mg each per 100 kg sea hare) and the consequent difficulties in purifying amounts sufficient for evaluation and use, have motivated efforts toward the synthesis of these compounds (Roux et al., Tetrahedron 50: 5345-5360 (1994); Shioiri et al., Tetrahedron 49: 1913-24 (1993); Patino et al., Tetrahedron 48: 4115-4122 (1992) and references cited therein). Synthetic Dolastatin 15, however, suffers from drawbacks which include poor solubility in aqueous systems and the need for expensive starting materials for its synthesis. These, in turn, have led to the synthesis and evaluation of structurally modified Dolastatin 15 derivatives [cf.: Biorg. Med. Chem. Lett. 4: 1947-50 (1994); WO 93 03054; JP-A-06234790; WO 93 23424].

However, there is a need for synthetic compounds with the biological activity of Dolastatin 15 which have useful aqueous solubility and can be produced efficiently and economically.

SUMMARY OF THE INVENTION

Compounds of the present invention include cell growth inhibitors which are peptides of Formula I,

A-B-D-E-F-(G),-(K)5-L

and acid salts thereof, wherein A, B, D, E, F, G and K are a-amino acid residues, and s and r are each, independently, 0 or 1. L is a monovalent radical, such as, for example, an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.

Another aspect of the present invention includes pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier. An additional embodiment of the present invention is a method for treating cancer in a mammal, such as a human, comprising administering to the manvnal an effective amount of a compound of Formula I in a pharmaceutically acceptable composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to peptides having antineoplastic activity. It also includes pharmaceutical compositions comprising these compounds and methods for treating cancer in a mammal, including a human, by administration of these compositions to the mammal.

Dolastatin 15, a peptide isolated from the sea hare Dolabella auricularia, is a potent inhibitor of cell growth. This compound, however, is present in trace quantities in the sea hare, and is thus difficult to isolate. Dolastatin 15 is also expensive to synthesize and suffers from poor aqueous solubility. As shown herein, however, Dolastatin 15 can serve as a starting point for the development of compounds which overcome these disadvantages while retaining antineoplastic activity or exhibiting greater antineoplastic activity than the natural product. Applicants have discovered that certain structural modifications of Dolastatin 15 provide compounds with a surprisingly improved therapeutic potential for the treatment of neoplastic diseases as compared to Dolastatin 10 and Dolastatin 15. Furthermore, the compounds of the present invention can be conveniently synthesized, as described below in detail.

For the purposes of the present invention, the term “monovalent radical” is intended to mean an electrically neutral molecular fragment capable of forming one covalent bond with a second neutral molecular fragment. Monovalent radicals include the hydrogen atom, alkyl groups, such as methyl, ethyl and propyl groups, halogen atoms, such as fluorine, chlorine and bromine atoms, aryl groups, such as phenyl and naphthyl groups, and alkoxy groups, such as methoxy and ethoxy groups. Two monovalent radicals on adjacent sigma-bonded atoms can also together form a pi bond between the adjacent atoms. Two monovalent radicals may also be linked together, for example, by a polymethylene unit, to form a cyclic structure. For example, the unit —N(R)R′, wherein R and R′ are each a monovalent radical, can, together with the nitrogen atom, form a heterocyclic ring. In addition, two monovalent radicals bonded to the same atom can together form a divalent radical, such as an oxygen atom or an alkylidene group, for example, a propylidene group.

For the purposes of the present invention, the term “normal alkyl” refers to an unbranched, or straight chain, alkyl group, for example, normal propyl (n-propyl, —CH₂CH₂ CH₃).

The compounds of the present invention can be represented by Formula I,

A-B-D-E-F-(G)_(r)-(K)_(s)-L  (I),

wherein A, B, D, E, F, G, and K are α-amino acid residues; s and r are each, independently, 0 or 1; and L is a monovalent radical such as an amino group, an N-substituted amino group, a β-hydroxylamino group, a hydrazido group, an alkoxy group, a thioalkoxy group, an aminoxy group, or an oximato group.

The peptides of Formula I are generally composed of L-amino acids but they can contain one or more D-amino acids. In the following discussion, reference to a particular amino acid includes both enantiomers unless a specific enantiomer is indicated. The present compounds can also be present as salts with physiologically-compatible acids, including hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.

The following is a description of the present invention, including a detailed description of individual components and of methods of using the claimed compounds.

Compounds of the Present Invention Identity of A

In one embodiment, A is a proline derivative of Formula II_(a),

where n, is an integer, preferably 0, 1, 2, or 3. R_(a) is a monovalent radical, such as a hydrogen atom or an unsubstituted or fluorine-substituted alkyl group, for example a normal, branched or cyclic C₁-C₃-alkyl group which is, optionally, substituted by from 1 to about 3 fluorine atoms; suitable examples include methyl, ethyl, isopropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, 1-methyl-2-fluoroethyl, 1-fluoromethyl-2-fluoroethyl or cyclopropyl; methyl, ethyl or isopropyl are preferred;

In this embodiment, R¹ _(a), is a monovalent radical, such as a hydrogen atom, an alkyl group, such as a methyl, ethyl or propyl group, or a phenyl group. The phenyl group can be substituted; suitable substituents include one or more halogen atoms, with fluorine, chlorine and bromine atoms preferred, C₁-C₄-alkyl groups, methoxy, ethoxy, trifluoromethyl or nitro groups. R_(a) and R¹ _(a) together can also form a propylene bridge.

R² _(a), R³ _(a), R⁴ _(a) and R⁵ _(a), are each, independently, a monovalent radical, such as a hydrogen atom or an alkyl, preferably, methyl, group.

In another embodiment, A is a substituted glycine derivative of Formula III_(a),

where R_(a) has the meaning stated for R_(a) in Formula II_(a) and, R¹ _(a), is a monovalent radical, for example, a hydrogen atom or a C₁-C₆-alkyl group, preferably a methyl, ethyl or propyl group.

In this embodiment, R⁶ _(a) is a monovalent radical, such as an alkyl, substituted alkyl, alkenyl, phenyl or substituted phenyl group. Suitable examples include methoxymethyl, 1-methoxyethyl, 1,1-dimethyl-hydroxymethyl, 1-trifluoromethylethyl, 1-trifluoromethyl-2,2,2-trifluoroethyl, vinyl, and 1-methylvinyl. Phenyl substituents can include one or more halogen atoms, preferably fluorine, chlorine or bromine atoms, and alkyl, methoxy, ethoxy, trifluoromethyl, and nitro groups.

When R¹ _(a) is an alkyl group, R⁶ _(a), can also be a C₁-C₆-alkyl, cycloalkyl, unsubstituted benzyl or substituted benzyl group. Suitable benzyl substituents include one or more halogen atoms, such as fluorine, chlorine or bromine atoms, C₁-C₄-alkyl groups, and methoxy, ethoxy, trifluoromethyl and nitro groups.

R⁷ _(a) is a monovalent radical, preferably a methyl, ethyl or isopropyl group.

In another embodiment, A is an α-amino acid derivative of Formula IV_(a),

where m_(a) is an integer, preferably 1 or 2, and R_(a) and R⁷ _(a) have the meanings stated for these substituents in Formula III_(a).

In another embodiment, A is an α-amino acid derivative of Formula V_(a),

where R_(a) and R⁷ _(a) have the meanings stated for R_(a) and R⁷ _(a), in Formula III_(a).

In a further embodiment, A is a substituted proline derivative of Formula VI_(a),

where R_(a) and R¹ _(a) have the meanings stated for R_(a) and R¹ _(a) in Formula II_(a), and X_(a) is a monovalent radical, preferably a hydroxyl, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.

In another embodiment, A is a thiaprolyl derivative of Formula VII_(a),

where R_(a), R¹ _(a), R² _(a), R³ _(a), R⁴ _(a) and R⁵ _(a) have the meanings stated for the respective substituents in Formula II_(a).

In another embodiment, A is a 1,3-dihydroisoindole derivative of Formula VIII_(a)

where R_(a) has the meaning stated for R_(a) for Formula II_(a).

In another embodiment, A is a 2-azabicyclo[2.2.1]heptane-3-carboxylic acid derivative of Formula IX_(a),

where Z_(a) is a single or double bond and R_(a) has the meaning stated for Formula II_(a). The 3-carbonyl substituent can have either the exo or endo orientation.

In another embodiment, A is an α-amino acid derivative of Formula X_(a),

where n_(a) has the meaning as stated for n_(a) for Formula II_(a), and R⁷ _(a), and R_(a) have the meanings as stated for R⁷ _(a) and R_(a) for Formula III_(a).

Identity of B

B is a valyl, isoleucyl, allo-isoleucyl, norvalyl, 2-tert-butylglycyl or 2-ethylglycyl residue. B can also be an α-amino acid residue of Formula II_(b),

in which R¹ _(b) and R² _(b) are each a monovalent radical. R¹ _(b) is, preferably, a hydrogen atom and R² _(b) is, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R² _(b) is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R¹ _(b) and R² _(b) together can be an isopropylidene group.

Identity of D

D is an N-alkylvalyl, N-alkyl-2-ethylglycyl, N-alkyl-2-tert-butylglycyl, N-alkyl-norleucyl, N-alkyl-isoleucyl, N-alkyl-allo-isoleucyl or N-alkyl-norvalyl residue, where the N-alkyl group is preferably a methyl group or an ethyl group.

In another embodiment, D is an α-amino acid residue of Formula II_(d),

where R_(d) has the meaning stated for R_(a) in Formula III_(a), R¹ _(d) is a monovalent radical, preferably a hydrogen atom, and R² _(d) is a monovalent radical, for example, an alkyl, alkoxyalkyl or alkenyl group. In preferred embodiments, R² _(d) is a cyclopropyl group, a normal or branched butyl, preferably tertiary-butyl, group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group such as a cyclopropyl group, a methoxymethyl group, a 1-methoxyethyl group or a 1-methylvinyl group. Additionally, R¹ _(d) and R² _(d) together can form an isopropylidene group.

Alternatively, D can be a proline derivative of Formula III_(d),

where n_(d) is an integer, for example, 1 or 2, and R³ _(b) has the meaning stated for R¹ _(a) in Formula III_(a). X_(d) is a monovalent radical, preferably a hydrogen atom, and, in the case where n_(d) equals 1, can also be a hydroxy or alkoxy, for example, methoxy or ethoxy, group or a fluorine atom.

Identity of E

E is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue, or a cyclic α-amino carboxylic acid residue of Formula II_(e),

where n_(e) is an integer, preferably 0, 1 or 2. R¹ _(e) has the meaning stated for R¹ _(a) in Formula III_(a). R² _(e), and R³ _(e) are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R⁴ _(e), is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R⁵ _(e), is a monovalent-radical, preferably a hydrogen atom or a fluorine atom. In the case where n_(e) is 1, R³ _(e) and R⁴ _(e) can together form a double bond, or R⁴ _(e), and R⁵ _(e) can together be a double-bonded oxygen radical. In the case where n_(e) has the value 1 or 2, R¹ _(e) and R² _(e) can together form a double bond.

In another embodiment, E is a 2- or 3-amino-cyclopentanecarboxylic acid residue of Formula III_(e),

where R_(e) is an alkyl group, such as methyl or ethyl, and R¹ _(e) has the meaning stated for R¹ _(a), in Formula III_(a).

Identity of F

F is a prolyl, thiazolidinyl-4-carbonyl, homoprolyl or hydroxyprolyl residue. F can also be a cyclic α-amino acid residue of Formula II,

where n_(f) is an integer, preferably 0, 1 or 2. R¹ _(f) has the meaning stated for R¹ _(a) in Formula III_(a). R² _(f) and R³ _(f) are each a monovalent radical, and can be, independently, a hydrogen atom or an alkyl, preferably methyl, group. R^(d) _(f) is a monovalent radical, preferably a hydrogen atom, a hydroxy, alkoxy, for example, methoxy or ethoxy, group or a fluorine atom. R⁵ _(f) is a monovalent radical, preferably a hydrogen atom or a fluorine atom. In the case where n_(r) has the value 1, R³ _(f) and R⁴ _(f) together can form a double bond or R⁴ _(f) and R⁵ _(f) can together be a double-bonded oxygen radical. In the case where n_(f) has the value 1 or 2, R¹ _(f) and R² _(f) can together form a double bond.

In another embodiment, F is a 2- or 3-amino-cyclopentanecarboxylic acid residue of Formula III_(f)

where R_(f) is a monovalent radical, such as a methyl or ethyl group, and R² _(f) has the meaning stated for R¹ _(a) in Formula II.

In another embodiment, F is an N-alkylglycyl or N-alkylalanyl residue, and the alkyl group is, preferably, a methyl group or an ethyl group.

Identity of G

G is an α-amino acid residue of Formula II_(g),

wherein R¹ _(g) is a hydrogen atom, or an alkyl group, for example, methyl, ethyl or n-propyl. R² _(g) can be, for example, a hydrogen atom, or an alkyl, arylalkyl, heteroarylalkyl or aryl group. Preferably, R² _(g) is an ethyl, isopropyl, tert-butyl, isobutyl, 2-methylpropyl, cyclohexylmethyl, benzyl, thiazolyl-2-methyl, pyridyl-2-methyl, n-butyl, 2,2-dimethylpropyl, naphthylmethyl, or n-propyl group, or a substituted or unsubstituted phenyl group. Suitable phenyl substituents include one or more halogen, preferably fluorine, chlorine or bromine, atoms, C₁-C₄-alkyl groups, methoxy, ethoxy, nitro or trifluoromethyl groups or a dioxomethylene group. Alternately, R¹ _(g) and R² _(g) can, together with the α-carbon atom, form a cyclopentane or cyclohexane ring or a benzo-fused cyclopentane ring, such as, for example, the indanyl group.

Identity of K

K is an α-amino acid residue of Formula II_(k),

wherein R¹ _(k) has the identity stated for R¹ _(g) in Formula II_(g), and R² _(k) has the identity stated for R² _(g) in Formula II_(g).

Identity of L

In one embodiment, L is an amino or substituted amino group of Formula II_(l),

where R¹ _(l) is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C₁-C₁₈-alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted aryl-C₁-C₆-alkoxy group, or a substituted or unsubstituted aryloxy-C₁-C₆— alkoxy or heteroaryl-C₁-C₆-alkoxy group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5- or 6-membered, preferably nitrogen-, oxygen- or sulfur-containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4- or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole, thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C₁-C₄-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.

R² _(l) is a monovalent radical, such as a hydrogen atom, a normal or branched, saturated or unsaturated C₁-C₁₈-alkyl group, a C₃-C₁₀-cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. The aryl group is preferably a phenyl or naphthyl group. The heteroaryl group is a 5- or 6-membered, preferably nitrogen-, oxygen- or sulfur-containing, ring system, such as, for example, a heteroaryl group derived from imidazole, isoxazole, isothiazole, thiazole, oxazole, pyrazole, thiophene, furan, pyrrole, 1,2,4- or 1,2,3-triazole, pyrazine, indole, benzofuran, benzothiophene, indole, isoindole, indazole, quinoline, pyridazine, pyrimidine, benzimidazole, benzopyran, benzothiazole, oxadiazole, thiadiazole or pyridine. Suitable aryl substituents include one or more halogen, preferably fluorine, bromine or chlorine, atoms, C₁-C₄-alkyl groups, methoxy, ethoxy or trifluoromethyl groups, a dioxymethylene group or nitro groups.

R² _(l) can, alternately, be of Formula II_(r),

where a_(l) is an integer, such as 0, 1, 2, 3, 4 or 5. R³ _(t) is a monovalent radical, preferably a lower alkyl group, such as a methyl, ethyl, propyl or isopropyl group. R⁴ _(l) is a monovalent radical, which can be a saturated or partially unsaturated carbocyclic system comprising from about 3 to about 10 carbon atoms, a substituted or unsubstituted aryl or heteroaryl group, with aryl and heteroaryl and preferred substituents having the meaning stated for R² _(l) in Formula II_(l).

R² _(l) can also be a substituent of Formula III_(r),

—(CH₂)₂—W_(l)—R⁵ _(l)  (III_(r)),

wherein W_(l) is an oxygen or sulfur atom or an N—R⁶ _(l) group. R⁵ _(l) is a monovalent radical, such as a hydrogen atom, a C₁-C₄-alkyl or C₃-C₇-cycloalkyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R² _(l) from Formula II_(l). R⁶ _(l) is a monovalent radical, preferably a hydrogen atom, a C₁-C₄-alkyl group or a C₃-C₇-cycloalkyl group, a C₁-C₁₈-alkanoyl group, a benzoyl group or a substituted or unsubstituted aryl or arylmethyl group, with aryl and its preferred substituents having the meaning stated for R² _(l) in Formula II_(l).

R² _(l) can, alternately, be a substituent of Formula IV_(r),

—(CH₂)_(b) _(l) —Z_(l)  (IV_(r)),

where b_(l) is an integer, preferably 2, 3 or 4. Z_(l) can be a monovalent radical such as a formyl, aminocarbonyl or hydrazinocarbonyl group, or a cyclic or acyclic acetal or thioacetal group.

R² _(l) can also be a substituent of Formula V_(r),

in which b_(l) has the above-mentioned meaning. R⁷ _(l) can be a monovalent radical, such as a polyglycol group of the formula —O—(CH₂—CH₂—O)_(d) _(l) —CH₃, where d_(l) is an integer; preferably in the range from about 2 to about 4 or from about 40 to about 90.

R² _(l) can further be a carbohydrate of Formula VI_(r),

where R² _(l) is a monovalent radical, such as a hydrogen atom, a C₁-C₄-alkanoyl or alkyl group, a benzoyl group or a benzyl group.

L can also be a β-hydroxylamino group of Formula III_(l),

where R² _(l) is a monovalent radical such as a hydrogen atom, a C₁-C₆-alkyl group or a substituted or unsubstituted aryl group, with aryl and its preferred substituents having the meaning stated for R² _(l)-R¹⁰ _(l) is a monovalent radical, preferably a hydrogen atom, alkyl, for example, methyl, or a phenyl group.

When r and/or s is l, L can also be an amino group of Formula IV_(l),

where R² _(l) and R⁴ _(l) are each a monovalent radical. R² _(l) and R⁴ _(l) can also be linked by a carbon-carbon bond.

Another subclass of compounds of this invention includes peptides of Formula I wherein L is a hydrazido group of Formula V_(l),

and R¹¹ _(l) is a monovalent radical, preferably a hydrogen atom. R¹² _(l) can be a monovalent radical such as a hydrogen atom, a normal or branched C₁-C₈-alkyl group, a C₁-C₃-cycloalkyl group, a C₃-C₈-cycloalkyl-C₁-C₄-alkyl group or a substituted or unsubstituted aryl, heteroaryl, aryl-C₁-C₄-alkyl or heteroaryl-C₁-C₄-alkyl group, where aryl, heteroaryl and their preferred substituents can be selected from among the options listed for R² _(l).

When r and/or s is l, R¹¹ _(l) can also be selected from among the options listed above for R¹² _(l), and the two radicals together can additionally form a propylene or butylene bridge.

Another subclass of compounds of this invention includes peptides of Formula I wherein L is a monovalent radical of the formula —O—R¹³ _(l) or the formula —S—R¹³ _(l), where R¹³ _(l) is a monovalent radical, such as a C₃-C₁₀-cycloalkyl group, a normal or branched C₂-C₁₆-alkenylmethyl group or a C₁-C₁₆-alkyl group which can be substituted by from 1 to about 5 halogen, preferably fluorine, atoms.

R¹³ _(l) can also be the radical —(CH₂)_(e)—R¹⁴ _(l), where e is an integer, preferably 1, 2 or 3. R¹⁴ _(l) is a monovalent radical, preferably a saturated or partially unsaturated C₃-C₁₁-carbocycle.

R¹³ _(l) can further be the monvalent radical —[CH₂—CH═C(CH₃)—CH₂]_(l)—H, where f is an integer, preferably 1, 2, 3 or 4.

R¹³ _(l) can also be the radical —[CH, —CH₂—O]_(g)—CH₃, where g is an integer, preferably in the range from 1 to about 5.

R¹³ _(l) can also be the radical —(CH₂)_(h)-aryl or —(CH₂)_(h)-heteroaryl, where aryl and heteroaryl can also be substituted and, along with their preferred substituents, can be selected from the group listed for R² _(l). h is an integer, preferably 0, 1, 2 or 3.

R¹³ _(l) can further be the radical —(CH₂)_(b)—W_(l)—R⁵ _(l). b, W_(l) and R⁵ _(l) can each be selected from among the options described for Formula IV_(l).

Another subclass of compounds of this invention includes peptides of Formula I in which L is an aminoxy group of the formula —O—N(R¹⁵ _(l))(R¹⁶ _(l)), where R¹⁵ _(l) and R¹⁶ _(l) are each a monovalent radical, which can independently be a hydrogen atom, a normal or branched C₁-C₈-alkyl group, which can be substituted by halogen, preferably fluorine, atoms, a C₃-C₈-cycloalkyl group, a C₃-C₈-cycloalkyl-C₁-C₄-alkyl group, a substituted or unsubstituted aryl or heteroaryl group or a substituted or unsubstituted aryl-C₁-C₄-alkyl group. Aryl and heteroaryl groups and the preferred substituents thereof can be selected from the options listed for R² _(l). R¹⁶ _(l) can be selected from among the options listed for R¹⁵ _(l). Additionally, R¹⁵ _(l) and R¹⁶ _(l) can together form a 5-, 6- or 7-membered heterocycle. The compounds of the present invention further comprise the salts of the compounds described above with physiologically tolerated acids.

Another subclass of compounds of this invention includes peptides of Formula I wherein L is an oximato group of the formula —O—N═C(R¹⁵ _(l))(R¹⁶ _(l)), R¹⁵ _(l) and R¹⁶ _(l) can be selected from among the options listed above and, additionally, can together form a cyclic system comprising, preferably, from about 3 to about 7 ring atoms. This cyclic system can additionally be fused to one or more aromatic rings. Particularly preferred cyclic systems are shown below.

In one embodiment, the invention provides compounds of Formula I wherein A is an amino acid derivative selected from among N-alkyl-D-prolyl, N-alkyl-L-prolyl, N-alkyl-D-piperidine-2-carbonyl, N-alkyl-L-piperidine-2-carbonyl, N,N-dialkyl-D-2-ethyl-2-phenylglycyl and N,N-dialkyl-L-2-ethyl-2-phenylglycyl, wherein alkyl is methyl, ethyl or isopropyl; and 13 is a valyl, isoleucyl or 2-t-butyl-L-glycyl residue.

Preferred compounds of the invention include compounds of Formula I wherein r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, L-N-methyl-piperidine-2-carbonyl, N,N-dimethylamino-iso-butyryl, N-methyl-L-prolyl, N-methyl-L-thiazolidine-4-carbonyl, N,N-dimethyl-glycyl, L-prolyl, L-piperidine-2-carbonyl, N-propyl-D-piperidine-2-carbonyl, D-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, D-prolyl, N-methyl-D-prolyl, N,N-dimethyl-2-(2-fluorophenyl)glycyl, 1-aza-[3,3,0]bicyclooctyl-5-carbonyl, N,N-dimethyl-2-[4-fluoro]phenyl-glycyl, N-methyl-[2,2,5,5-tetrameramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 2-[N,N-dimethylamino]indane-2-carbonyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, N-isopropyl-2-(R,S)-ethyl-2-phenylglycyl, 1-[N,N-dimethyl-amino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-2-[4-methoxy]phenyl-glycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methylpiperidine-2-carbonyl, N-methyl-L-prolyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, N,N-dimethyl-[O-methyl]threonyl, N-methyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl, 1-[N,N-dimethylamino]cyclopentyl-1-carbonyl and 1,2,3,4-tetrahydroisoquinoline-3-carbonyl. B is valyl, isoleucyl or 2-tert-butylglycyl. D is N-methylvalyl, N-methyl-2-t-butylglycyl or N-methylisoleucyl. E and F are each, independently, prolyl, thiaprolyl, homoprolyl, hydroxyprolyl, 3,4-didehydroprolyl, 4-fluoroprolyl, and 3-methylprolyl. L is an alkoxy group or an amino group of the formula R¹ _(l)—N—R² _(l), wherein R¹ _(l) and R² _(l) are independently selected from the group consisting of hydrogen, alkoxy, hydroxy, alkyl and alkylaryl.

In a particularly preferred subset of the compounds of the invention, r and s are each 0. A is an amino acid derivative selected from among D-N-methyl-piperidine-2-carbonyl, N-ethyl-D-piperidine-2-carbonyl, N-isopropyl-D-piperidine-2-carbonyl, N,N-dimethyl-2-cyclopropyl-glycyl, N-methyl-D-prolyl, L-aza-[3,3,0]bicyclooctyl-5-carbonyl, N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carbonyl, 2-(R,S)-ethyl-2-phenylglycyl, D,L-1-aminoindane-1-carbonyl, N,N-dimethyl-2-(R,S)-methyl-2-phenylglycyl, 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carbonyl, 1-[N,N-dimethylamino]indane-2-carbonyl, N,N-dimethyl-2-propyl-2-phenylglycyl, N,N-dimethyl-L-2-ethyl-2-phenylglycyl, N,N-dimethyl-D-2-ethyl-2-phenylglycyl, N-methyl-3-hydroxy-D,L-valyl, N,N-dimethyl-D,L-2-isopropyl-2-phenylglycyl, N-methyl-piperidine-2-carbonyl, N-methyl-D,L-prolyl, N-methyl-1,2,3,4-tetra-hydroisoquinoline-1-carbonyl, N-methylazetidine-2-carbonyl, N-isopropylazetidine-2-carbonyl, N,N-dimethyl-[O-methyl]seryl, 1-[N,N-dimethylamino]cyclohexyl-1-carbonyl and L-[N,N-dimethylamino]cyclopentyl-1-carbonyl. B is valyl; D is N-methylvalyl; and E and F are each prolyl. L is a C₁-C₆-alkoxy group or an amino group of the formula R¹ _(l)—N—R² _(l), wherein R¹ _(l) and R² _(l) are each independently selected from the group consisting of hydrogen, C₁-C₆-alkoxy, hydroxy, normal, cyclic or branched C₁-C₁₂-alkyl, and phenylalkyl.

Synthetic Methods

The compounds of the present invention can be prepared by known methods of peptide synthesis. Thus, the peptides can be assembled sequentially from individual amino acids or by linking suitable small peptide fragments. In sequential assembly, the peptide chain is extended stepwise, starting at the C-terminus, by one amino acid per step. In fragment coupling, fragments of different lengths can be linked together, and the fragments in turn can be obtained by sequential assembly from amino acids or by fragment coupling of still shorter peptides.

In both sequential assembly and fragment coupling it is necessary to link the units by forming an amide linkage, which can be accomplished via a variety of enzymatic and chemical methods. Chemical methods for forming the amide linkage are described in detail in standard references on peptide chemistry, including Miller, Methoden der organischen Chemie Vol. XV/2, 1-364, Thieme Verlag, Stuttgart, (1974); Stewart and Young, Solid Phase Peptide Synthesis, 31-34 and 71-82, Pierce Chemical Company, Rockford, Ill. (1984); Bodanszky et al., Peptide Synthesis, 85-128, John Wiley & Sons, New York, (1976). Preferred methods include the azide method, the symmetric and mixed anhydride method, the use of in situ generated or preformed active esters, the use of urethane protected N-carboxy anhydrides of amino acids and the formation of the amide linkage using coupling reagents, such as carboxylic acid activators, especially dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), pivaloyl chloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N,N-bis(2-oxo-oxazolidinyl)amidophosphoryl chloride (BOP—Cl), bromo-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBrop), diphenyl-phosphoryl azide (DPPA), Castro's reagent (BOP, PyBop), O-benzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HBTU), O-azabenzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HATU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich's reagent; HOTDO), and 1,1-carbonyldi-imidazole (CDI). The coupling reagents can be employed alone or in combination with additives such as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxyazabenzotriazole (HOAt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.

Although the use of protecting groups is generally not necessary in enzymatic peptide synthesis, reversible protection of reactive groups not involved in formation of the amide linkage is necessary for both reactants in chemical synthesis. Three conventional protective group techniques are preferred for chemical peptide synthesis: the benzyloxycarbonyl (Z), the t-butoxycarbonyl (Boc) and the 9-fluorenylmethoxy-carbonyl (Fmoc) techniques. Identified in each case is the protective group on the α-amino group of the chain-extending unit. A detailed review of amino-acid protective groups is given by Müller, Methoden der organischen Chemie Vol. XV/1, pp 20-906, Thieme Verlag, Stuttgart (1974). The units employed for assembling the peptide chain can be reacted in solution, in suspension or by a method similar to that described by Merrifield, J. Am. Chem. Soc. 85: (1963) 2149.

Solvents suitable for peptide synthesis include any solvent which is inert under the reaction conditions, especially water, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of these solvents.

Peptide synthesis on the polymeric support can be carried out in a suitable inert organic solvent in which the amino acid derivatives starting materials are soluble. However, preferred solvents additionally have resin-swelling properties, such as DMF, DCM, NMP, acetonitrile and DMSO, and mixtures of these solvents. Following synthesis, the peptide is removed from the polymeric support. The conditions under which this cleavage is accomplished for various resin types are disclosed in the literature. The cleavage reactions most commonly used are acid- or palladium-catalyzed, the former being conducted in, for example, liquid anhydrous hydrogen fluoride, anhydrous trifluoromethanesulfonic acid, dilute or concentrated trifluoroacetic acid, and acetic acid/dichloromethane/trifluoroethanol mixtures. The latter can be carried out in THF or THF-DCM-mixtures in the presence of a weak base, such as morpholine. Certain protecting groups are also cleaved off under these conditions.

Partial deprotection of the peptide may also be necessary prior to certain derivatization reactions. For example, peptides dialkylated at the N-terminus can be prepared by coupling the appropriate N,N-di-alkylamino acid to the peptide in solution or on the polymeric support, by reductive alkylation of the resin-bound peptide in DMF/1% acetic acid with NaCNBH₃ and the appropriate aldehyde or by hydrogenation of the peptide in solution in the presence of the appropriate aldehyde or ketone and Pd/carbon.

The various non-naturally occurring amino acids as well as the various non-amino acid moieties disclosed herein can be obtained from commercial sources or synthesized from commercially available staring materials using methods known in the art. For example, amino acid building blocks with R¹ and R² groups can be prepared according to the method described by Wuensch and Weyl, Methoden der Organische Chemie, vol. XV, Springer Verlag: Stuttgart, p. 306 (1974) and references cited therein.

Methods of Use of the Claimed Compounds

In another embodiment, the present invention comprises a method for partially or totally inhibiting formation of, or otherwise treating (e.g., reversing or inhibiting the further development of) solid tumors (e.g., tumors of the lung, breast, colon, prostate, bladder, rectum, or endometrial tumors) or hematological malignancies (e.g., leukemias, lymphomas) in a mammal, for example, a human, by administering to the mammal a therapeutically effective amount of a compound or a combination of compounds of Formula I. The compound(s) may be administered alone or in a pharmaceutical composition comprising the compound(s) and an acceptable carrier or diluent. Administration can be by any of the means which are conventional for pharmaceutical, preferably oncological, agents, including oral and parenteral means, such as subcutaneously, intravenously, intramuscularly and intraperitoneally, nasally or rectally. The compounds may be administered alone or in the form of pharmaceutical compositions containing a compound or compounds of Formula I together with a pharmaceutically accepted carrier appropriate for the desired route of administration. Such pharmaceutical compositions may be combination products, i.e., they may also contain other therapeutically active ingredients.

The dosage to be administered to the mammal, such as a human, will contain a therapeutically effective amount of a compound described herein. As used herein, “therapeutically effective amount” is an amount sufficient to inhibit (partially or totally) formation of a tumor or a hematological malignancy or to reverse development of a solid tumor or other malignancy or prevent or reduce its further progression. For a particular condition or method of treatment, the dosage is determined empirically, using known methods, and will depend upon factors such as the biological activity of the particular compound employed; the means of administration; the age, health and body weight of the recipient; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies; and the effect desired. A typical daily dose will be from about 0.05 to about 50 milligrams per kilogram of body weight by oral administration and from about 0.01 to about 20 milligrams per kilogram of body weight by parenteral administration.

The compounds of the present invention can be administered in conventional solid or liquid pharmaceutical administration forms, for example, uncoated or (film-) coated tablets, capsules, powders, granules, suppositories or solutions. These are produced in a conventional manner. The active substances can for this purpose be processed with conventional pharmaceutical aids such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, sustained release compositions, antioxidants and/or propellant gases (cf. H. Sücker et al.: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1978). The administration forms obtained in this way typically contain from about 1 to about 90% by weight of the active substance.

The present invention will now be illustrated by the following examples, which are not limiting.

EXAMPLES

The proteinogenous amino acids are abbreviated in the examples using the known three-letter code. Other abbreviations employed are: TFA=trifluoroacetic acid, Ac=acetic acid, DCM=dichloromethane, DMSO=dimethylsulfoxide, Bu=butyl, Et=ethyl, Me=methyl, Bzl=benzyl. In the compounds listed, all proteinogenous amino acids are L-amino acids unless otherwise noted. Other abbreviations used: Me₂Val=N,N-dimethylvaline, MeVal=N-methylvaline, Bn=benzyl, Me₂Aib=[2-N,N-dimethylamino]-isobutyric acid.

General Procedures

The peptides of the invention are synthesized either by classical solution synthesis using standard Z- and Boc-methodology as described above or by standard methods of solid-phase synthesis using Boc and Fmoc protective group techniques.

In the case of solid phase synthesis, the N,N-dialkyl-penta- or hexapeptide acids are liberated from the solid support and further coupled with the corresponding C-terminal amines in solution. BOP—Cl and PyBrop were used as reagents for coupling of the amino acid following the N-methylamino acids. The reaction times were correspondingly increased. For reductive alkylation of the N-terminus, the peptide-resin was deprotected at the N terminus and then reacted with a 3-fold molar excess of aldehyde or ketone in DMF/1% acetic acid with addition of 3 equivalents of NaCNBH₃. After the reaction was complete (negative Kaiser test) the resin was washed several times with water, isopropanol, DMF and dichloromethane.

In solution synthesis, the use of either Boc-protected amino acid NCAs (N-tert.-butyloxycarbonyl-amino acid-N-carboxy-anhydrides), Z-protected amino acid NCAs (N-benzyloxycarbonyl-amino acid-N-carboxy-anhydrides), or the use of pivaloyl chloride as condensing agent respectively is most advantageous for coupling of the amino acid following the N-methylamino acids. Reductive alkylation of the N terminus can e.g. be achieved by reaction of the N-terminally deprotected peptides or amino acids with the corresponding aldehydes or ketones using NaCNBH₃ or hydrogen-Pd/C.

Valyl-N-methylvalyl-prolyl-prolylbenzylamide hydrochloride for example was prepared according to methods disclosed in German Patent Application No. DE 19527575 A1.

Purification and Characterization of the Peptides

Peptide purification was carried out by gel chromatography (SEPHADEX G-10, G-15/10% HOAc, SEPHADEX LH₂0/MeOH), medium pressure chromatography (stationary phase: HD-SIL C-18, 20-45 micron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1% TFA/water), preparative HPLC (stationary phase: Waters Delta-Pak C-18, 15 micron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1% TFA/water), or by crystallization.

The purity of the resulting products was determined by analytical HPLC (stationary phase: 100 2.1 mm VYDAC C-18, 5 micron, 300 A; mobile phase: acetonitrile-water gradient, buffered with 0.1% TFA, 40° C.; or 3.9 mm VYDAC C-18, 30° C.). Characterization was by fast atom bombardment mass spectroscopy and NMR-spectroscopy.

Example 1 Synthesis of [N-Methyl-L-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn (Compound 1) and [N-Methyl-D-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn (Compound 2) Preparation of N-methyl-piperidine-2-carboxylic acid

N-Methyl-piperidine-2-carboxylic acid ethyl ester (5.1 g) was dissolved in a mixture of 100 ml methanol and 10 ml water. NaOH (8 g) was added and the reaction mixture was stirred at room temperature overnight. The solution was then neutralized with hydrochloric acid, evaporated to dryness, and evaporated four times with toluene. The resulting powdery residue was used directly in the next step.

Preparation of [N-Methyl-piperidine-2-carbonyl]-Val-MeVal-Pro-Pro-NHBn

The residue prepared as described above (5.05 g) and H-Val-MeVal-Pro-Pro-NHBn×HCl (4.88 g) were dissolved in 50 ml dry DMF. After cooling the solution in an ice bath, 1.52 g DEPCN and 2.66 ml triethylamine were added. The reaction mixture was stirred at 0° C. for 2 h and then at room temperature overnight. The DMF was removed by evaporation under reduced pressure. The residue was diluted with dichloromethane and the organic phase was washed with aqueous hydrochloric acid (pH 2) and water, dried over sodium sulfate and evaporated to dryness. The diastereomeric mixture was then separated by flash chromatography with a gradient using heptane/ethyl acetate and dichloromethane/methanol. Under the HPLC conditions described in the previous section (C-18 reverse phase) isomer 1 has a retention time of 14.9 minutes, and isomer 2 has a retention time of 15.8 minutes. Both isomers were characterized by fast atom bombardment mass spectrometry ([M+H]+=639).

Example 2 Preparation of Me₂Aib-Val-MeVal-Pro-Pro-NHBn (Compound 3) Preparation of 2-[N,N-dimethylamino]-isobutyric acid

2-Amino-isobutyric acid (10.3 g) was dissolved in 200 ml methanol. After addition of 25 ml aqueous formaldehyde and 1 g 10% Pd/C, the reaction mixture was hydrogenated overnight at room temperature. The catalyst was filtered, and the filtrate was evaporated to dryness. The residue was crystallized from isopropanal to give 4.8 g of the desired product.

Preparation of Me₂Aib-Val-MeVal-Pro-Pro-NHBn×HCl

2-[N,N-Dimethylamino]-isobutyric acid (1.3 g, 10 mmol) and 5.5 g (10 mmol) H-Val-MeVal-Pro-Pro-NHBn×HCl were dissolved in 50 ml dry DMF. After cooling to 0° C., 1.6 g DEPCN (10 mmol) and 2.9 ml triethylamine were added to the reaction mixture. The resulting mixture was stirred at 0° C. for 2 h and at room temperature overnight. Ice water (50 mL) was then added, and the resulting mixture was extracted twice with diethyl ether. The ether extracts were washed with 1 N NaOH (1×) and aqueous NaCl (3×), then dried over sodium sulfate and evaporated to dryness under reduced pressure. The product was crystallized from 100 ml diethyl ether with HCl/ether, and recrystallized from acetone to give 1.2 g of the desired product, which was characterized by fast atom bombardment mass spectrometry ([M+H]+=627).

Example 3 Preparation of [N,N-dimethyl-2-ethyl-2-phenylglycyl]-Val-MeVal-Pro-Pro-NHBn×HCl (Compound 4) Preparation of [N,N-dimethyl-2-ethyl-2-phenylglycyl]-Val-MeVal-Pro-Pro-NHBn×HCl

2.07 g (10 mmol) N,N-Dimethyl-2-ethyl-2-phenylglycine and 5.5 g (10 mmol) H-Val-MeVal-Pro-Pro-NHBn×HCl were dissolved in 100 ml dry DMF. After cooling to 0° C., 1.6 g DEPCN (10 mmol) and 2.9 ml triethylamine were added. The reaction mixture was stirred at 0° C. for 2 h and at room temperature overnight, then worked up as described above. The crude product was crystallized from diethyl ether with HCl/ether to give 4 g of the desired product, which was characterized by fast atom bombardment mass spectrometry ([M+H]+=703).

Example 4 Preparation of [N-Methyl-D-Pro]-Val-MeVal-Pro-Pro-NHBn (Compound 5) Preparation of Z-D-Pro-Val-MeVal-Pro-Pro-NHBn

3.74 g Z-D-Pro-OH (15 mmol, BACHEM) and 8.25 g H-Val-MeVal-Pro-Pro-NHBn×HCl (15 mmol) were dissolved in 80 ml dry DMF. After cooling to 0° C., 2.4 g DEPCN (2.25 ml, 15 mmol) and 4.2 ml triethylamine (30 mmol) were added. The reaction mixture was stirred at 0° C. for several hours and room temperature overnight, then the DMF was evaporated under reduced pressure. The residue was diluted with ethyl acetate and thoroughly washed with dilute aqueous HCl (pH 2), water, dilute aqueous NaOH (pH 9-10), and water. The organic phase was dried over sodium sulfate and evaporated to dryness to yield 9.2 g of the desired protected pentapeptide.

Preparation of D-Pro-Val-MeVal-Pro-Pro-NHBn×HCl

8.2 g (11 mmol) Z-D-Pro-Val-MeVal-Pro-Pro-NHBn was dissolved in 70 ml methanol. After addition of 0.7 ml concentrated hydrochloric acid and 0.3 g 10% Palladium/charcoal to the solution, the resulting mixture was hydrogenated. Filtration and evaporation of the solvent gave a residue which was dissolved in water, adjusted to pH 2 and extracted twice with ethyl acetate. The aqueous phase was adjusted to pH 9-10 and extracted twice with dichloromethane. The organic extracts were evaporated and the residue was redissolved in diethylether and crystallized by addition of HCl/ether as the hydrochloride salt to give 6.5 g of the desired product.

Preparation of [N-methyl-D-Pro]-Val-MeVal-Pro-Pro-NHBn×HCl

1.94 g (3 mmol) of D-Pro-Val-MeVal-Pro-Pro-NHBn×HCl was dissolved in 30 ml methanol. To this solution was then added 0.3 g 10% Pd/charcoal and 1.5 ml aqueous formaldehyde solution and the reaction mixture was hydrogenated. Following filtration and evaporation of the solvents, the resulting residue was dissolved in water, adjusted to pH 2 and extracted twice with diethyl ether and several additional times with dichloromethane. The aqueous phase was adjusted to pH 9-10 and extracted twice with dichloromethane. The organic extracts were dried over sodium sulfate and evaporated to dryness. The residue was crystallized as the hydrochloride salt to give 0.5 g of the desired product which was characterized by fast atom bombardment mass spectrometry ([M+H]+=625).

The compounds listed in Table 1 were prepared according to the methods described in Examples 1-4. Where compounds are referred to as “isomer 1” or “isomer 2”, isomer 1 is the diastereomer with the shorter retention time on the reversed phase analytical HPLC system. Fast atom bombardment-mass spectrometry results for selected compounds are provided in Table 2.

TABLE 1 Compound No. Compound 6 Xah Val Xaa Pro Xab 7 Xai Val Xaa Pro Xab 8 Xae Val Xaa Pro Xab 9 Xad Val Xaa Pro Xbr 10 Xam Val Xaa Pro Xab 11 Xaw Ile Xaa Pro Xbx 12 Xao Val Xaa Pro Xab 13 Xad Val Xaa Pro Xap 14 Xaq Val Xaa Pro Xab 15 Xar Val Xaa Pro Xab 16 Xas Val Xaa Pro Xab 17 Xat Val Xaa Pro Xab isomer 1 18 Xat Val Xaa Pro Xab isomer 2 19 Xaf Val Xaa Pro Xab 20 Xav Val Xaa Pro Xab 21 Xag Val Xaa Pro Xab 22 Xax Val Xaa Pro Xab isomer 1 23 Xax Val Xaa Pro Xab isomer 2 24 Xay Val Xaa Pro Xab 25 Xaz Val Xaa Pro Xab isomer 1 26 Xaz Val Xaa Pro Xab isomer 2 27 Xba Val Xaa Pro Xab 28 Xbb Val Xaa Pro Xab 29 Xbc Val Xaa Pro Xab 30 Xbd Val Xaa Pro Xab isomer 1 31 Xbd Val Xaa Pro Xab isomer 2 32 Xbe Val Xaa Pro Xab isomer 1 33 Xbe Val Xaa Pro Xab isomer 2 34 Xbf Val Xaa Pro Xab isomer 1 35 Xbg Val Xaa Pro Xab 36 Xbh Val Xaa Pro Xab isomer 1 37 Xbh Val Xaa Pro Xab isomer 2 38 Xbi Val Xaa Pro Xab isomer 1 39 Xbi Val Xaa Pro Xab isomer 2 40 Xbk Val Xaa Pro Xab isomer 1 41 Xbk Val Xaa Pro Xab isomer 2 42 Xbl Val Xaa Pro Xab 43 Xbf Val Xaa Pro Xab isomer 2 44 Xbm Val Xaa Pro Xab 45 Xaw Val Xaa Pro Xbn 46 Xbo Val Xaa Pro Xbn isomer 1 47 Xbo Val Xaa Pro Xbn isomer 2 48 Xaw Val Xaa Pro Xbp 49 Xbo Val Xaa Pro Xbp isomer 1 50 Xbo Val Xaa Pro Xbp isomer 2 51 Xaw Val Xaa Pro Xbq 52 Xaw Val Xaa Pro Xbr 53 Xbs Val Xaa Pro Xbt isomer 1 54 Xbl Val Xaa Pro Xab isomer 1 55 Xbl Val Xaa Pro Xab isomer 2 56 Xbu Val Xaa Pro Xab isomer 1 57 Xbv Val Xaa Pro Xab 58 Xbw Val Xaa Pro Xab isomer 1 59 Xbw Val Xaa Pro Xab isomer 2 60 Xbs Val Xaa Pro Xbt isomer 2 61 Xbu Val Xaa Pro Xab isomer 2 62 Xbo Val Xaa Pro Xbr isomer 1 63 Xbo Val Xaa Pro Xbr isomer 2 64 Xbo Val Xaa Pro Xbq isomer 1 65 Xbo Val Xaa Pro Xbq isomer 2 66 Xaw Val Xaa Pro Xbx 67 Xby Val Xaa Pro Xab 68 Xbz Val Xaa Pro Xab 69 Xca Val Xaa Pro Xab isomer 1 70 Xca Val Xaa Pro Xab isomer 2 71 Xbo Val Xaa Pro Xbx isomer 1 72 Xbo Val Xaa Pro Xbx isomer 2 73 Xau Val Xaa Pro Xbp 74 Xau Val Xaa Pro Xbx 75 Xbi Val Xaa Pro Xbx isomer 2 76 Xau Val Xaa Pro Xab isomer 1 77 Xau Val Xaa Pro Xab isomer 2 78 Xau Val Xaa Pro Xcb 79 Xbi Val Xaa Pro Xcb isomer 1 80 Xbi Val Xaa Pro Xcb isomer 2 81 Xbi Val Xaa Pro Xcc isomer 1 82 Xbi Val Xaa Pro Xcc isomer 2 83 Xbi Val Xaa Pro Xcd 84 Xbk Val Xaa Pro Xcc isomer 1 85 Xbk Val Xaa Pro Xcc isomer 2 86 Xax Val Xaa Pro Xbp isomer 1 87 Xax Val Xaa Pro Xbp isomer 2 88 Xbk Val Xaa Pro Xcb isomer 1 89 Xbk Val Xaa Pro Xcb isomer 2 90 Xau Val Xaa Pro Xcc 91 Xau Val Xaa Pro Xcd 92 Xba Val Xaa Pro Xcb isomer 1 93 Xba Val Xaa Pro Xcb isomer 2 94 Xbo Val Xaa Pro Xbp isomer 1 95 Xbo Val Xaa Pro Xbp isomer 2 96 Xau Val Xaa Pro Xbp isomer 1 97 Xau Val Xaa Pro Xbp isomer 2 98 Xbi Val Xaa Pro Xcd isomer 2 99 Xbk Val Xaa Pro Xcd 100 Xba Val Xaa Pro Xbp isomer 1 101 Xba Val Xaa Pro Xbp isomer 2 102 Xba Val Xaa Pro Xcc isomer 1 103 Xba Val Xaa Pro Xcc isomer 2 104 Xba Val Xaa Pro Xcd 105 Xce Val Xaa Pro Xab 106 Xcf Val Xaa Pro Xab 107 Xcg Val Xaa Pro Xab isomer 1 108 Xcg Val Xaa Pro Xab isomer 2 109 Xaw Val Xaa Pro Xch 110 Xaw Val Xaa Pro Xci 111 Xaw Val Xaa Pro Xck 112 Xaw Val Xaa Pro Xcl 113 Xaw Val Xaa Pro Xcm 114 Xaw Val Xaa Pro Xcn 115 Xaw Val Xaa Pro Xco 116 Xaw Val Xaa Pro Xcp 117 Xaw Val Xaa Pro Xcq 118 Xaw Val Xaa Pro Xcr 119 Xad Val Xaa Pro Xch 120 Xad Val Xaa Pro Xci 121 Xad Val Xaa Pro Xck 122 Xad Val Xaa Pro Xcl 123 Xad Val Xaa Pro Xcm 124 Xad Val Xaa Pro Xcn 125 Xad Val Xaa Pro Xco 126 Xad Val Xaa Pro Xcp 127 Xad Val Xaa Pro Xcq 128 Xad Val Xaa Pro Xcr 129 Xad Val Xaa Pro Xbx 130 Xau Val Xaa Pro Xch 131 Xau Val Xaa Pro Xci 132 Xau Val Xaa Pro Xck 133 Xau Val Xaa Pro Xcl 134 Xau Val Xaa Pro Xcm 135 Xau Val Xaa Pro Xcn 136 Xau Val Xaa Pro Xco 137 Xau Val Xaa Pro Xcp 138 Xau Val Xaa Pro Xcq 139 Xau Val Xaa Pro Xcr 140 Xau Val Xaa Pro Xbr 141 Xad Val Xaa Xal Xbx 142 Xau Val Xaa Xal Xbx 143 Xaw Val Xaa Xal Xbx 144 Xad Val Xaa Xal Xch 145 Xau Val Xaa Xal Xch 146 Xaw Val Xaa Xal Xch 147 Xad Val Xaa Xal Xcr 148 Xau Val Xaa Xal Xcr 149 Xaw Val Xaa Xal Xcr 150 Xad Val Xaa Xan Xbx 151 Xau Val Xaa Xan Xbx 152 Xaw Val Xaa Xan Xbx 153 Xad Val Xaa Xan Xch 154 Xau Val Xaa Xan Xch 155 Xaw Val Xaa Xan Xch 156 Xad Val Xaa Xan Xcr 157 Xau Val Xaa Xan Xcr 158 Xaw Val Xaa Xan Xcr 159 Xau Ile Xaa Pro Xbx 160 Xad Ile Xaa Pro Xbx 161 Xaw Ile Xaa Pro Xch 162 Xad Ile Xaa Pro Xch 163 Xau Ile Xaa Pro Xch 164 Xaw Xcs Xaa Pro Xch 165 Xad Xcs Xaa Pro Xch 166 Xau Xcs Xaa Pro Xch 167 Xaw Xcs Xaa Pro Xbx 168 Xad Xcs Xaa Pro Xbx 169 Xau Xcs Xaa Pro Xbx 170 Xaw Val Xct Pro Xch 171 Xad Val Xct Pro Xch 172 Xau Val Xct Pro Xch 173 Xaw Val Xct Pro Xbx 174 Xad Val Xct Pro Xbx 175 Xau Val Xct Pro Xbx The symbols Xaa in Table 1 represent the following amino acids or residues thereof:

Xaa: N-methyl-valine Xab: Prolyl N-benzylamide

Xac: L-N-methyl-piperidine-2-carboxylic acid Xad: D-N-methyl-piperidine-2-carboxylic acid

Xae: N-methyl-L-proline

Xaf: N-methyl-L-thiazolidine-4-carboxylic acid

Xag: N,N-dimethylglycine Xah: L-proline

Xai: L-piperidine-2-carboxylic acid Xak: 2-[N,N-dimethylamino]-isobutyric acid Xal: L-thiazolidine-4-carboxylic acid Xam: N-propyl-D-piperidine-2-carboxylic acid Xan: L-3,4-didehydroproline Xao: D-piperidine-2-carboxylic acid Xap: proline tert.butylester Xaq: N-ethyl-D-piperidine-2-carboxylic acid Xar: N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carboxylic acid Xas: N-isopropyl-D-piperidine-2-carboxylic acid Xat: N,N-dimethyl-2-cyclopropyl-glycine Xau: N,N-dimethyl-2-ethyl-2-phenyl-glycine

Xav: D-proline

Xaw: N-methyl-D-pro line Xax: N,N-dimethyl-2-[2-fluoro]phenyl-glycine Xay: 1-aza-[3,3,0]bicyclooctyl-5-carboxylic acid Xaz: N,N-dimethyl-2-[4-fluoro]phenyl-glycine Xba: N-methyl-[2,2,5,5-tetramethyl]-thiazolidine-2-carboxylic acid Xbb: 2-(R,S)-ethyl-2-phenyl-glycine Xbc: D,L-1-aminoindane-1-carboxylic acid Xbd: N,N-dimethyl-2-(R,S)-methyl-2-phenyl-glycine Xbe: 2-[N,N-dimethylamino]indane-2-carboxylic acid Xbf: 5-[N,N-dimethylamino]-5,6,7,8-tetrahydro-naphthalene-5-carboxylic acid Xbg: N-isopropyl-2-(R,S)-ethyl-2-phenyl-glycine Xbh: 1-[N,N-dimethylamino]indane-2-carboxylic acid Xbi: N,N-dimethyl-2-propyl-2-phenyl-glycine Xbk: N,N-dimethyl-2-[4-methoxy]phenyl-glycine Xbl: N-methyl-3-hydroxy-D,L-valine Xbm: N,N-dimethyl-D,L-2-isopropyl-2-phenyl-glycine Xbn: proline-N-methoxy-N-methyl-amide Xbo: N-methyl-piperidine-2-carboxylic acid Xbp: proline-isopropylamide Xbq: proline-isoxazolidinyl Xbr: proline-N-methoxy-N-benzylamide

Xbs: N-methyl-D,L-proline

Xbt: proline-[5-phenyl]isoxazolidinyl Xbu: N-methyl-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid Xbv: N-methyl-azetidine-2-carboxylic acid Xbw: N-isopropyl-azetidine-2-carboxylic acid Xbx: proline-tert-butylamide Xby: N,N-dimethyl-[O-methyl]serine Xbz: N,N-dimethyl-[O-methyl]threonine Xca: N-methyl-1,2,3,4-tetrahydro isoquino line-3-carboxylic acid Xcb: proline-pentyl(3)amide Xcc: proline-(R)-phenethylamide Xcd: proline-(S)-phenethylamide Xce: 1-[N,N-dimethylamino]cyclohexyl-1-carboxylic acid Xcf: 1-[N,N-dimethylamino]cyclopentyl-1-carboxylic acid Xcg: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

Xch:

Xci:

Xck:

Xcl:

Xcm:

Xcn:

Xco:

Xcp:

Xcq: N

Xcr:

Xcs: L-2-tert-butyl-glycine

Xct: N-methyl-L-Isoleucine

TABLE 2 Results of FAB-MS analysis of selected compounds Compound No. Mol. weight measured 1 639 2 639 3 627 4 703 5 625 6 611 7 625 8 625 10 667 12 625 13 606 14 653 15 699 16 667 17 639 18 639 19 643 20 611 21 599 22 693 23 693 24 651 25 693 26 693 27 699 28 675 29 673 30 689 31 689 32 701 33 701 34 715 35 717 36 701 37 701 38 717 39 717 40 705 41 705 42 643 43 715 44 703 45 579 46 593 47 593 48 577 49 591 50 591 51 591 52 655 53 667 54 657 55 657 56 687 57 611 58 639 59 639 60 667 61 687 62 669 63 669 64 605 65 605 66 591 67 643 68 657 69 687 70 687 71 605 72 605 73 655 74 669 75 683 76 703 77 703 78 683 79 697 80 697 81 731 82 731 83 731 84 719 85 719 86 645 87 645 88 685 89 685 90 717 91 717 92 679 93 679 94 591 95 591 96 655 97 655 98 731 99 719 100 651 101 651 102 713 103 713 104 713 105 666 106 653 107 687 108 687

Example 5 Evaluation of Biological Activity In Vitro Methodology

Cytotoxicity was measured using a standard methodology for adherent cell lines, such as the microculture tetrazolium assay (MTT). Details of this assay have been published (Alley, M. C. et al., Cancer Research 48: 589-601, (1988)). Exponentially growing cultures of HT-29 colon carcinoma cells were used to make microtiter plate cultures. Cells were seeded at 5000-20,000 cells per well in 96-well plates (in 150 mL of media), and grown overnight at 37° C. Test compounds were added, in 10-fold dilutions varying from 10⁻⁴ M to 10⁻¹⁰ M. Cells were then incubated for 48 hours. To determine the number of viable cells in each well, the MTT dye was added (50 mL of a 3 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in saline). This mixture was incubated at 37° C. for 5 hours, and then 50 mL of 25% SDS, pH 2, was added to each well. After an overnight incubation, the absorbance of each well at 550 nm was read using an ELISA reader. The values for the mean+/−SD of data from replicated wells were calculated, using the formula % T/C (% viable cells treated/control). The concentration of test compound which gives a T/C of 50% growth inhibition was designated as the IC₅₀.

Table 3 presents the IC₅₀ values determined in the HT-29 assay for a series of compounds of the invention.

TABLE 3 Compound No. HT-29 [IC₅₀] 1 4.7 × 10⁻⁸ 2  6.8 × 10⁻¹⁰ 3 3.5 × 10⁻⁸ 4 1.2 × 10⁻⁹ 5 5.0 × 10⁻⁹ 8 5.1 × 10⁻⁷ 10 1.3 × 10⁻⁷ 12 3.7 × 10⁻⁷ 13 1.0 × 10⁻⁹ 14 1.5 × 10⁻⁹ 15 1.7 × 10⁻⁷ 16  7.3 × 10⁻¹⁰ 17 6.3 × 10⁻⁸ 18 8.8 × 10⁻⁹ 22 6.4 × 10⁻⁷ 24 2.8 × 10⁻⁸ 27 3.7 × 10⁻⁸ 28 4.9 × 10⁻⁸ 29 3.6 × 10⁻⁸ 30 6.1 × 10⁻⁹ 31 2.0 × 10⁻⁷ 32 8.5 × 10⁻⁷ 33 1.2 × 10⁻⁶ 34 5.0 × 10⁻⁹ 35 1.4 × 10⁻⁷ 36 6.2 × 10⁻⁹ 37 1.9 × 10⁻⁷ 38 7.3 × 10⁻⁷ 39 2.5 × 10⁻⁸ 40 5.6 × 10⁻⁷ 41 7.3 × 10⁻⁶ 42 3.4 × 10⁻⁷ 43 5.9 × 10⁻⁸ 44 4.8 × 10⁻⁸ 45 5.6 × 10⁻⁸ 46 7.2 × 10⁻⁷ 47 2.3 × 10⁻⁸ 48 2.5 × 10⁻⁸ 49 8.8 × 10⁻⁸ 50 8.9 × 10⁻⁸ 51 4.6 × 10⁻⁸ 52 3.4 × 10⁻⁷ 53 5.0 × 10⁻⁹ 54 4.2 × 10⁻⁹ 55 5.6 × 10⁻⁸ 57 2.5 × 10⁻⁸ 58 6.3 × 10⁻⁸ 59 1.9 × 10⁻⁷ 60 1.8 × 10⁻⁹ 62 9.9 × 10⁻⁸ 63 5.6 × 10⁻⁸ 64 1.7 × 10⁻⁶ 65 9.7 × 10⁻⁸ 66 3.4 × 10⁻⁷ 67 3.4 × 10⁻⁷ 68 4.2 × 10⁻⁷ 70 7.1 × 10⁻⁶ 72 1.2 × 10⁻⁷ 73 1.4 × 10⁻⁹ 74 5.1 × 10⁻⁸ 75 8.5 × 10⁻⁷ 76  2.3 × 10⁻¹⁰ 77 7.2 × 10⁻⁹ 78 4.3 × 10⁻⁹ 79 1.7 × 10⁻⁶ 80 6.7 × 10⁻⁸ 81 1.3 × 10⁻⁷ 82 1.1 × 10⁻⁸ 83 1.3 × 10⁻⁷ 84 1.2 × 10⁻⁶ 85 9.5 × 10⁻⁶ 90  9.3 × 10⁻¹⁰ 91  8.3 × 10⁻¹⁰ 92 1.5 × 10⁻⁶ 93 1.8 × 10⁻⁶ 94 3.0 × 10⁻⁶ 95 1.1 × 10⁻⁸ 96 1.7 × 10⁻⁹ 97 3.2 × 10⁻⁸ 98 6.0 × 10⁻⁹ 99 3.8 × 10⁻⁶ 100 2.3 × 10⁻⁶ 101 2.1 × 10⁻⁶ 102 1.2 × 10⁻⁷ 103 1.1 × 10⁻⁷ 104 3.5 × 10⁻⁶ 105 1.8 × 10⁻⁸ 106 9.7 × 10⁻⁸ 108 7.1 × 10⁻⁶

In Vivo Methodology

Compounds of this invention may be further tested in any of the various preclinical assays for in vivo activity which are indicative of clinical utility. Such assays are conducted with nude mice into which tumor tissue, preferably of human origin, has been transplanted (“xenografted”), as is well known in this field. Test compounds are evaluated for their anti-tumor efficacy following administration to the xenograft-bearing mice.

More specifically, human tumors grown in athymic nude mice can be transplanted into new recipient animals, using tumor fragments which are about 50 mg in size. The day of transplantation is designated as day O, Six to ten days later, the mice are treated with the test compounds given as an intravenous or intraperitoneal injection, in groups of 5-10 mice at each dose. Compounds are given daily for 5 days, 10 days or days, at doses from 10-100 mg/kg body weight. Tumor diameters and body weights are measured twice weekly. Tumor masses are calculated using the diameters measured with Vernier calipers, and the formula:

(length×width²)/2=mg of tumor weight

Mean tumor weights are then calculated for each treatment group, and T/C values are determined for each group relative to the untreated control tumors.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the following claims. 

1-39. (canceled)
 40. A compound of the formula A-B-D-E-F-(G)_(r)-(K)_(s)-L wherein r and s are 0; A is a proline derivative of Formula II_(a),

wherein n_(a) is 0 to 2; R_(a) is hydrogen or unsubstituted normal or branched C₁-C₃-alkyl; R¹ _(a) is hydrogen; or R_(a) and R¹ _(a) together form a propylene bridge; and R² _(a), R³ _(a), R⁴ _(a), and R⁵ _(a) are each hydrogen; B is valyl, isoleucyl, or 2-tert-butylglycyl; D is N-methylvalyl or N-methylisoleucyl; E is prolyl, thiazolidinyl-4-carbonyl or 3,4-didehydroprolyl; F is prolyl; and L is N-benzylamide, N-methoxy-N-benzylamide, tert-butylamide, tert-butyl ester, N-methoxy-N-methylamide, isopropylamide, 5-phenyl isoxasolidinyl, isoxazolidinyl, pentylamide, (R) or (S) phenethylamide, —O-tert-butyl, isoxazolidinyl, 5-phenyl isoxazolidinyl,

or a pharmaceutically acceptable salt thereof.
 41. A compound of claim 40 selected from the group consisting of: Xad Ile Xaa Pro Xbx Xad Ile Xaa Pro Xch Xad Val Xaa Pro Xap Xad Val Xaa Pro Xbr Xad Val Xaa Pro Xbx Xad Val Xaa Pro Xch Xad Val Xaa Pro Xci Xad Val Xaa Pro Xck Xad Val Xaa Pro Xcl Xad Val Xaa Pro Xcm Xad Val Xaa Pro Xcn Xad Val Xaa Pro Xco Xad Val Xaa Pro Xcp Xad Val Xaa Pro Xcq Xad Val Xaa Pro Xcr Xad Val Xaa Xal Xbx Xad Val Xaa Xal Xch Xad Val Xaa Xal Xcr Xad Val Xaa Xan Xbx Xad Val Xaa Xan Xch Xad Val Xaa Xan Xcr Xad Val Xct Pro Xbx Xad Val Xct Pro Xch Xad Xcs Xaa Pro Xbx Xad Xcs Xaa Pro Xch Xae Val Xaa Pro Xab Xah Val Xaa Pro Xab Xai Val Xaa Pro Xab Xam Val Xaa Pro Xab Xao Val Xaa Pro Xab Xaq Val Xaa Pro Xab Xas Val Xaa Pro Xab Xav Val Xaa Pro Xab Xaw Ile Xaa Pro Xbx Xaw Ile Xaa Pro Xch Xaw Val Xaa Pro Xbn Xaw Val Xaa Pro Xbp Xaw Val Xaa Pro Xbq Xaw Val Xaa Pro Xbr Xaw Val Xaa Pro Xbx Xaw Val Xaa Pro Xch Xaw Val Xaa Pro Xci Xaw Val Xaa Pro Xck Xaw Val Xaa Pro Xcl Xaw Val Xaa Pro Xcm Xaw Val Xaa Pro Xcn Xaw Val Xaa Pro Xco Xaw Val Xaa Pro Xcp Xaw Val Xaa Pro Xcq Xaw Val Xaa Pro Xcr Xaw Val Xaa Xal Xbx Xaw Val Xaa Xal Xch Xaw Val Xaa Xal Xcr Xaw Val Xaa Xan Xbx Xaw Val Xaa Xan Xch Xaw Val Xaa Xan Xcr Xaw Val Xct Pro Xbx Xaw Val Xct Pro Xch Xaw Xcs Xaa Pro Xbx Xaw Xcs Xaa Pro Xch Xbo Val Xaa Pro Xbn isomer 1 Xbo Val Xaa Pro Xbn isomer 2 Xbo Val Xaa Pro Xbp isomer 1 Xbo Val Xaa Pro Xbp isomer 1 Xbo Val Xaa Pro Xbp isomer 2 Xbo Val Xaa Pro Xbp isomer 2 Xbo Val Xaa Pro Xbq isomer 1 Xbo Val Xaa Pro Xbq isomer 2 Xbo Val Xaa Pro Xbr isomer 1 Xbo Val Xaa Pro Xbr isomer 2 Xbo Val Xaa Pro Xbx isomer 1 Xbo Val Xaa Pro Xbx isomer 2 Xbs Val Xaa Pro Xbt isomer 1 and Xbs Val Xaa Pro Xbt isomer 2 or a pharmaceutically acceptable salt thereof, wherein: Xaa is N-methyl-valine, Xab is Prolyl N-benzylamide, Xad is D-N-methyl-piperidine-2-carboxylic acid, Xae is N-methyl-L-proline, Xah is L-proline, Xai is L-piperidine-2-carboxylic acid, Xak is 2-[N,N-dimethylamino]-isobutyric acid, Xal is L-thiazolidine-4-carboxylic acid, Xam is N-propyl-D-piperidine-2-carboxylic acid, Xan is L-3,4-didehydroproline, Xao is D-piperidine-2-carboxylic acid, Xap is proline tertbutylester, Xaq is N-ethyl-D-piperidine-2-carboxylic acid, Xar is N-methyl-[2,2,5,5-tetramethyl]-L-thiazolidine-2-carboxylic acid, Xas is N-isopropyl-D-piperidine-2-carboxylic acid, Xav is D-proline, Xaw is N-methyl-D-proline, Xax is N,N-dimethyl-2-[2-fluoro]phenyl-glycine, Xbn is proline-N-methoxy-N-methyl-amide, Xbo is N-methyl-piperidine-2-carboxylic acid, Xbp is proline-isopropylamide, Xbq is proline-isoxazolidinyl, Xbr is proline-N-methoxy-N-benzylamide, Xbs is N-methyl-D,L-proline, Xbt is proline-[5-phenyl]isoxazolidinyl, Xbx is proline-tert-butylamide, Xch is

Xci is

Xck is

Xcl is

Xcm is

Xcn is

Xco is

Xcp is

Xcq is

Xcr

Xcs is L-2-tert-butyl-glycine, and Xct is N-methyl-L-isoleucine. 